The complete chloroplast genome of an Antarctic moss, Ptychostomum pseudotriquetrum (Hedw.) J.R.Spence & H.P.Ramsay (Bryaceae), and phylogenetic analysis

Abstract Ptychostomum pseudotriquetrum (Hedw.) J.R.Spence & H.P.Ramsay (Bryaceae) is a bipolar and one of the most widespread species within Antarctica, exhibiting a ubiquitous presence along the Antarctic Peninsula. This study analyzed its chloroplast genome, which is 123,172 bp in length, and consists of 82 protein-coding genes, four ribosomal RNA genes, and 31 transfer RNA genes. A phylogenetic tree, constructed using 58 conserved orthologous protein-coding genes from 19 complete chloroplast genomes of the class Bryopsida, confirmed that P. pseudotriquetrum belongs to clade Bryaceae. Within this clade, P. pseudotriquetrum diverged from the clade containing Anomobryum gemmigerum and Bryum argenteum. This study contributes to enriching chloroplast genome resources for the family Bryaceae and the genus Ptychostomum. Such advancement could facilitate future genetic investigations aimed at conserving and exploiting Antarctic bryophytes.


Introduction
Ptychostomum pseudotriquetrum (Hedw.)J.R.Spence & H.P.Ramsay, known by its homotypic synonyms Bryum pseudotriquetrum (Hedw.)P.Gaertn., B.Mey. & Scherb.and Plagiobryum pseudotriquetrum (Hedw.)N.Pedersen, belongs to the family Bryaceae (Holyoak and Pedersen 2007).While P. pseudotriquetrum is often regarded as a cosmopolitan species, it is more precisely described as a bipolar moss with intermediate stations found at high elevations in tropical Africa.Within Antarctica, it ranks among the most widespread moss species, exhibiting a ubiquitous presence throughout the South Orkney Islands and South Shetland Islands archipelagos, with varied distribution along the Antarctic Peninsula.Furthermore, this species is a common component of the Antarctic herb tundra formation, often coexisting with the only two species of vascular plants native to this biome, Deschampsia antarctica and Colobanthus quitensis (Ochyra et al. 2008).
It typically thrives in well-drained, sheltered habitats, predominantly on sloping ground at low elevations in coastal areas.In Antarctica, certain populations of P. pseudotriquetrum are submerged in lakes, occasionally reaching considerable depths.Records of P. pseudotriquetrum growth have been documented in freshwater lakes, such as Radok Lake in continental Antarctica, where it has been observed at depths of up to 81 m (Wagner and Seppelt 2006).
Given the morphological variations observed as inconsistent phenotypes of P. pseudotriquetrum, taxonomic inference is derived not only from the assessment of morphological traits but also from phylogenetic analyses of DNA sequences across various phenotypes of this species (Holyoak and Heden€ as 2006).Furthermore, investigations into the flavonoid composition of P. pseudotriquetrum (referred to as Bryum algens) by Webby et al. (1996) have uncovered multiple chemotypes within this species.The current study aims to enhance the understanding of the taxonomic position of P. pseudotriquetrum and its evolution within clade Bryaceae by analyzing its chloroplast genome, a subject not previously explored.
Total genomic DNA was purified using a BiomedicVR Plant gDNA Extraction Kit (Biomedic Co., Ltd., Bucheon, South Korea) according to the manufacturer's protocol.Genomic library construction was performed using a TruSeq PCR Free DNA Sample Prep Kit (Illumina, San Diego, CA), and pairedend whole-genome sequences were produced using the Illumina NovaSeq platform (San Diego, CA).A total of 14,813,700 filtered reads with a mean length of 145.0 bp were obtained, and the total read length of raw data was 2.4 Gb.The raw sequencing data underwent trimming followed by de novo assembly with CLC Assembly Cell v4.2.1 (CLC bio, Aarhus, Denmark).Among the assembled contigs, chloroplast genome sequences were retrieved, aligned, and merged into a single sequence using the Sanionia uncinata chloroplast genome sequence as a reference (Park et al. 2018).The assembly underwent manual verification to finalize it, and any sequence errors were corrected through read mapping against the assembled contig.To verify the accuracy of the assembly, trimmed raw sequence data were mapped to the assembled chloroplast genome and the average coverage depth was �720.6 (Supplementary Figure 1).
having two introns.Ten cis-spliced genes (atpF, clpP, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, and ycf3) and one transspliced gene (rps12) were verified to be corrected and annotated with multiple sequence alignment (Supplementary Figure 2).To evaluate the evolutionary relationships, 19 chloroplast genome sequences including four complete chloroplast genomes of Bryaceae were used.As shown in Figure 3, Bryaceae and Mniaceae are clustered together to form the order Bryales, which is consistent with a previous study on the phylogenetics of the Bryophyte (Cole et al. 2019).

Figure 1 .
Figure 1.Reference images of Ptychostomum pseudotriquetrum.It is one of the most widespread moss species found in Antarctica, typically growing in moist and relatively sheltered locations such as the edges of meltwater streams (A).The characteristic morphological feature is the dense extensive carpets of small-sized plants with whitish uppermost leaves (B).Photographs were taken by Hyoungseok Lee in Barton Peninsula, King George Island (62 � 13 0 49.9 00 S; 58 � 42 0 40.3 00 W) on 18 February 2022.

Figure 2 .
Figure 2. Map of the chloroplast genome of Ptychostomum pseudotriquetrum.Genes lying outside the outer circle are transcribed clockwise, while those inside the circle are transcribed counterclockwise.Genes belonging to different functional groups are color-coded.The innermost darker grey corresponds to GC content, while the lighter grey corresponds to at content.IR: inverted repeat; LSC: large single-copy region; SSC: small single-copy region.